Enhancing quinoa growth under severe saline-alkali stress by phosphate solubilizing microorganism Penicillium funicuiosum P1

Promoting the growth of plants and improving plant stress-resistance by plant growth-promoting microorganism increasingly become a hotpot. While, most researchers focus on their supply role of nutrition or plant hormone. In this study, a novel mechanism that phosphate solubilizing microorganisms promoted plant growth under saline-alkali stress through secretion of organic acids, was proposed. The effects of desulfurization gypsum, humic acid, organic fertilizer and phosphate-solubilizing microorganism Penicillium funicuiosum P1 (KX400570) on the growth of quinoa (Chenopodium quinoa cv. Longli 1), showed that the survival rate, stem length and dry weight of quinoa treated with P1 were 2.5, 1.5, 1 and 1.5 times higher than those of sterile water (CK) under severe saline-alkali stress. The growth-promoting effect of P1 on quinoa was much better than that of other treatment groups. In addition, P1 promoted the growth of quinoa because the organic acids (malic acid, citric acid, succinic acid, etc.) from P1 stimulated the antioxidant system and promote the photosynthesis of quinoa, further promote quinoa growth.

To reduce the stress of saline-alkali soil for plant, the researchers presented lots of methods, including physical [8], chemical [9] and biological methods [10]. Among of them, physical methods, especially salt washing method, worked best [11]. While, its complex operation and high cost hindered their large-scale application [11]. Comparatively, chemical methods were the most common methods for improving saline-alkali soil because of their simple operation and low cost. However, chemical methods such as desulfurization gypsum [12] and humic acid [13], did not work in a short-term [12][13][14]. The biological methods, including plant of pioneer crops [15], application of microorganism agent [16][17][18] and organic fertilizer, etc. a1111111111 a1111111111 a1111111111 a1111111111 a1111111111

Materials source
Phosphate-solubilizing fungi Penicillium funicuiosum P1 (KX400570) was preserved in our lab, and the organic acids secreted by P1 were determined qualitatively and quantitatively by liquid chromatograph Ultimate3000 [45]. The organic fertilizer was also prepared ourselves [46]. Quinoa used in this paper was Chenopodium quinoa cv. Longli 1, which was bred by Pasture and Green Agriculture Institute of Gansu Academy of Agricultural Sciences. And the quinoa seeds were bought from Shanxi Jinnongyuan Land Products Co., Ltd.. Desulfurized gypsum and humic acid were purchased from Liyuan Environmental Protection Materials Co., Ltd. and Shenzhen Dugao New Biological Technology Co., Ltd., respectively.

Plant growth conditions and treatment
Pot experiments. The saline-alkali soil used in pot experiments was collected from the surface 0-20 cm depth of Liujia saline-alkali soil (44˚13 ' 59.559'E, 125˚6 ' 41.349'N) in Nong ' an County, Changchun City, Jilin Province, China. The soil was dried and separated by a 0. 9 mm sieve, and then put into no-hole pots. The maximum, minimum diameter and height of the pots are 18.5 cm, 10 cm and 11cm, respectively. After that, different treatments were applied to the soil, including sterile water (CK), desulfurized gypsum (FGD), commercial amendments (MA, humic acid content � 70%, 3% of the soil), organic fertilizer (OF, 3% of the soil) and phosphate-solubilizing fungi P1 (P1, 20 mL bacterial solution per 1 kg soil after PDA culture), respectively. Quinoa seeds sterilized were planted in pots (1 kg soil per pot, 35 seeds per pot, 3 replicates). The plants were placed in a greenhouse with a room temperature of 25˚C, and the light / dark cycle was 16 h / 8 h. Samples were tested after 30 days. And each test was repeated triple.
Soilless culture experiment. Quinoa seeds sterilized were seeded in a 200 mL container with vermiculite. Each container was input suitable Na 2 CO 3 -NaHCO 3 buffer solution (0.1 M Na 2 CO 3 : 0.1 M Na 2 HCO 3 = 5: 5) and Hoagland solution. Then five exogenous organic acids were added to the system, which were oxalic acid, acetic acid, succinic acid, citric acid and malic acid. The type and concentration of these organic acids were determined by the secretion of P1. And then 10 quinoa seeds were planted in each container. Each treatment has three parallel samples.

Biochemical analysis
Chlorophyll content of quinoa was determined by portable chlorophyll meter (TYS-4N, Beijing Zhongkeweihe Instrument Company, China). Three mature leaves of three quinoa plants were selected, and the average value of each leaf was recorded three times in vivo.
Proline content was tested by the Acidic Ninhydrin Method [47]. 0.5 g of plant leaves were added into 5 mL 3% sulfosalicylic acid solution, and then heated in boiling water for 10 minutes. After that, 2 mL centrifuged supernatant was mixed with 2 mL acetic acid and 2 mL acidic ninhydrin reagent. Then, it was heated in boiling water bath for 30 min. Then, the solution was cooled, then mixed with 4 mL toluene and stood. Finally, the upper solution was taken and tested at 520 nm wavelength by L5S BMS (INESA Analytical Instrument Co., Ltd), and proline content was got from a standard curve prepared using analytical grade proline and expressed as mg/g FW.
Malondialdehyde (MDA) content was tested by thiobarbituric acid (TBA) method. 0.1 g plant samples were added to 5 mL 5% trichloroacetic acid (TCA) solution, and then ground, homogenized and centrifuged. After that, 2 mL supernatant was taken and mixed with 2 mL 0.67% thiobarbituric acid (TBA). Then, the mixing solution was heated in boiling water bath for 30 min, and following with the measurement of absorbance values at 450 nm, 532 nm and 660 nm by L5S BMS (INESA Analytical Instrument Co., Ltd). MDA content was expressed on a nmol/g FW basis [48].
Antioxidant enzyme (POD, SOD) activity was detected by Zhou W et al. [49] method. In the SOD experiments, 0.1 g plant samples were grinded with liquid nitrogen, and then washed with 5 mL phosphate buffer (pH 7.8). After centrifugation, the crude enzyme solution was got. Then, 0.05 g crude enzyme solution reacted with SOD reaction solution, under 4000lx light intensity for 20 min. And then, the absorbance was measured at 560 nm by L5S BMS (INESA Analytical Instrument Co., Ltd). SOD reaction solution was prepared by 0.05 mol/L phosphate buffer (pH 7.8), 130 mmol/L methionine solution, 750 μmol/L nitro-blue tetrazolium solution, 100 μmol/L EDTA-Na 2 solution and 20 μmol/L riboflavin solution, according to the ratio of 5: 1: 1: 1: 1. In the POD experiment, 0.1 g plant samples were grinded with liquid nitrogen, and then washed with 5 mL phosphate buffer (pH 5.5). After centrifugation, the crude enzyme solution was got. Then, 0.1 mL crude enzyme solution was added to POD reaction solution, and then the absorbance was measured at 470 nm by L5S BMS (INESA Analytical Instrument Co., Ltd). POD reaction solution was prepared with 2.9 mL phosphate buffer (pH 5.5), 1.0 mL 2% hydrogen peroxide and 1.0 mL 0.05 mol/L guaiacol, according to the ratio of 5: 1: 1: 1: 1. Each test was repeated triple.

Soil physical and chemical properties
Soil physical and chemical properties include pH, electrical conductivity, HCO 3 content, organic matter, available nitrogen, available phosphorus and available potassium. Soil pH and electrical conductivity were measured after mixing the soil with deionized water at a ratio of 1: 5 by pH meter (SevenDirect SD20 HA Kit, METTLER TOLEDO company) and conductivity meter (DDS-11A, Shanghai INESA Scientific Instrument Co, Ltd.), respectively [24]. Soil organic matter were tested by K 2 Cr 2 O 7 -H 2 SO 4 digestion method [50]. 0.5 g soil sample was mixed with 1 mol/L K 2 Cr 2 O 7 solution. Then, concentrated sulfuric acid and o-phenanthroline indicator was added into the reaction system. And then, the solution was titrated with 0.5 mol/L FeSO 4 till brick red. Finally, record the consumption of FeSO 4 and calculate the content of organic matter through the consumption.
Available nitrogen was tested by alkali-diffusion method [51]. 2.0 g soil sample was evenly mixed with 10 mL 1 mol/L NaOH in the external chamber of the diffusion dish, and then 2 mL boric acid indicator was put into the internal chamber. Then, the reaction was done at 40˚C for 24 h. After that, the reaction solution was titrated with 0.01 mol/L H 2 SO 4 till yellow. Finally, record the consumption of H 2 SO 4 and calculate the content of organic matter through the consumption.
Available phosphorus was tested by molybdenum antimony colorimetric method [52]. 2.5 g soil samples were added in 0. 5 mol/L NaHCO 3 to extract available phosphorus, and then the filtrate was reacted with molybdenum antimony reagent for 30 min, and read absorbance at 880 nm by L5S BMS (INESA Analytical Instrument Co., Ltd). Then, available phosphorus concentration was determined according to the standard curve.
Available potassium was tested by flame photometry [53]. Available potassium was extracted from 5.0 g soil sample by neutral NH 4 OAc solution. Then, the soil filtrate and a series of potassium standard solutions were determined by 6400A flame photometer (INESA Analytical Instrument Co., Ltd. And then, standard curve was established, and potassium concentration in filtrate was determined according to standard curve. Each test was repeated triple.

Statistical analysis
One-way ANOVA were determined using SPSS Statistics 25 (IBM, New York, USA). The PCoA was used to express the impact of different treatment methods on soil physical and chemical properties and plants by principal coordinates using a Bray-Curtis dissimilarity matrix calculated from the taxonomic abundance matrices. The PCoA plots were generated using the vegan package [54] in R studio (version 4.1.1).

Results and discussion
Salinity-alkali stress seriously inhibits the growth of plants [5,55]. Generally, amendments are applied to improve saline-alkali soil under saline-alkali stress [9, 13, 39, 56]. However, chemical amendments work little on plant growth in a short term under saline-alkali stress [9, 14]. From Table 1, although the MDA content ( Fig 1A) and proline content ( Fig 1B) were decreased after the application of desulfurization gypsum and humic acid, the growth of quinoa seedlings were not significantly improved. The results have the same tendency with that of Yonggan Zha et al. [12] and Hu, Y. W. et al. [13]. While, organic fertilizer works better on quinoa's growth. After organic fertilizer treatment, quinoa survival rate, root and stem length, fresh and dry weight, were increased by about 2 times, 60% and 1 times compared with CK, respectively (Table 1). Moreover, the content of both MDA ( Fig 1A) and proline ( Fig 1B) decreased by about 65%. This indicated that organic fertilizer treatment not only alleviated saline-alkali stress, but also effectively promoted quinoa growth. Why did organic fertilizer improve quinoa growth? Recent studies provided possible answers: organic fertilizers were beneficial for optimizing saline-alkali soil microbial community [20,38]. Organic fertilizer provided nutrients for microorganisms and then increased the abundance of beneficial microorganisms [20,57]. And then, thanks to these beneficial microorganisms, organic fertilizer plays a role on assisting plants to resist saline-alkali stress [19,58]. In another words, beneficial microorganisms were able to help plants resist saline-alkali stress [34]. Then, will it be better if we apply beneficial microorganisms directly to the saline-alkali soil?
To solve the problem, we studied the effect of phosphate-solubilizing microorganism P1 on assisting quinoa to resist saline-alkali stress and its mechanism. As shown in Table 1, the treatment inoculated with P1 better promote the growth of quinoa under severe saline-alkali stress than organic fertilizer. The survival rate of quinoa increased by about 20% (Table 1). Both fresh weight and dry weight of quinoa increased by about 10% (Table 1). Furthermore, compared with organic fertilizer, MDA ( Fig 1A) and proline (Fig 1B) content of quinoa decreased by 52% and 21%, respectively. Thus, beneficial microorganisms were more favorable for promoting plant growth.
In summary, soil amendments were beneficial for promoting the growth of quinoa under saline-alkali stress. Among them, microbial agent P1 worked best. P1 increased the survival rate, root and stem length, fresh and dry weight per plant by 2.5 times, 1.5 times, 55%, 1 time and 1.5 times, respectively, under severe saline-alkali stress. The experimental results were consistent with those of Lei Kong et al. [59] and Mehrnoush Eskandari Torbaghan et al. [60]. Lei Kong et al. [59] inoculated AMF fungi into saline-alkali soil. And the stem length of tomato was increased by 39.32%. But, P1 increased the stem length of quinoa by 55%. Mehrnoush Eskandari Torbagha et al. [60] inoculated Alkalibacillus haloalkaliphilus in saline-alkali soil. And the fresh weight of wheat was increased by about 70%. But, P1 doubled the fresh weight of quinoa. What caused the remarkable effect of phosphate-solubilizing microorganisms P1 on quinoa?
Previous researches illustrated that phosphate-solubilizing microorganisms can improve soil properties and provide suitable growth environment for plant growth [61][62][63]. Fungi P1 had similar function as well. P1 secreted organic acids during their growth process. These organic acids decreased soil pH and bicarbonate concentration (Table 2). But improvement of soil properties was impossible for the reason of quinoa growth. From the statistical analysis,

PLOS ONE
Enhancing quinoa growth under severe saline-alkali stress by microorganism organic fertilizer treatment performed best on improving saline-alkali soil properties, followed by humic acid, as shown in Fig 2. However, the treatment with P1 works the best on the growth of quinoa (Fig 3). This phenomenon indicated that some important factors were ignored. Then, whether organic acids secreted by P1 not only reduced the saline-alkali soil pH, but also promoted plant growth? Some exogenous organic acids promoted the growth of plants. Some exogenous organic acids such as succinic acid increased organic acids (succinic acid, citric acid, malic acid and oxalic acid, etc.) in plant cytoplasm [64]. The increase of intracellular organic acid content was beneficial for plants to resist saline-alkali stress especially oxalic acid [65], and then resulted in promoting plant growth [66,67]. Does P1 secret similar organic acids? In order to solve this problem, we detected the organic acids secreted by P1. As shown in Table 3, P1 secretes a variety of organic acids, among which oxalic acid, acetic acid, citric acid, malic acid and succinic acid are abundant. Therefore, we studied the effects of exogenous oxalic acid, acetic acid, citric acid, malic acid and succinic acid on the growth of quinoa, respectively (Fig 4). The results   showed that all these acids had obvious growth-promoting effects on quinoa under different pH buffer conditions. Moreover, the growth-promoting effect of organic acids on plants increased with the increase of pH. Among them, oxalic acid, acetic acid and malic acid had better growth-promoting effect on quinoa. In pH 9 buffer solution, the stem length and root length of quinoa in oxalic acid treatment group increased by 1.5 and 3 times, respectively. Both stem and root length of quinoa in the acetic acid and malic acid treatment groups both increased by about 1.5 times.
Organic acids promoted plant growth may due to the following two reasons. Firstly, organic acids activated the antioxidant system of plants under stress, and improved the tolerance of plants to abiotic stress. Thus, plant growth was improved. Succinic acid induced the accumulation of succinic acid, citric acid, malic acid and oxalic acid in plants, up-regulated the gene expression of malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC) in plant roots, and effectively alleviated aluminum stress and promote the growth of plants [64]. Oxalic acid and citric acid improved the SOD and POD activities of Larix olgensis and activated the antioxidant system of the plants under lead stress and improved the growth of Larix olgensis [68]. Citric acid and acetic acid improved the SOD and POD activities of Melilotus officinalis under copper stress, and improved the growth of Melilotus officinalis [69]. From here we see that the activation of antioxidant system can effectively assist plants to resist stress and promote plant growth. Similar results were obtained in this experiment. The organic acids secreted by P1 also actived the antioxidant system, and increased SOD ( Fig 5A) and POD (Fig 5B) in quinoa by 30% and 16%, respectively. Secondly, organic acids promoted photosynthesis of plants. Malic acid enhanced photosynthesis by increasing chlorophyll content [70] or inducing stomatal opening [71]. Acetic acid also enhanced photosynthesis by increasing chlorophyll content [72]. Citric acid enhanced photosynthesis by increasing carotenoid content and the number and volume of chloroplasts in mesophyll cells [70]. Similar phenomenon was observed in our experiment. The chlorophyll content in quinoa treated with P1 was significantly higher than other treatments. (Fig 6) Therefore, organic acids promoting photosynthesis of quinoa is another reason why these organic acids promote quinoa growth. It can be found that the organic acids secreted by P1 played an important role on assisting quinoa to resist saline-alkali stress and promoting its growth.
In summary, the main reason why phosphate-solubilizing microorganisms promote plant growth is that they secrete organic acids. On the one hand, these organic acids reduced soil pH and provided a better growth environment for plants. On the other hand, they affect the metabolic system of plants, improve the resistance capability of plants and promote photosynthesis.

Conclusion
Different methods of alleviating saline alkali stress have great differences in promoting plant growth, due to different mechanisms. Although the improvement effect on soil was obvious after the application of chemical amendments, the growth of quinoa was only improved little. However, biological methods not only worked on improving saline-alkali soil properties, but also promoted quinoa growth. Both organic fertilizer and P1 microbial agent activated the antioxidant system of quinoa. And the organic acids secreted by P1 not only played a role on soil properties improvement, but also were used as bioactive substances to promote quinoa. We believe that small molecular organic acids which have biological activity secreted by microorganisms play a very important role on plant resistance to stress. In the future, P1 agent is going to applied in open field experiments and the effect of P1 on other plants, such as rice, wheat, corn and so on, will be studied as well. Furthermore, the effect of P1 on soil microorganism community and saline-alkali stress response gene express of plant are going to study.